It will be important to understand DENV serotype-specific immunodominance hierarchy of TS and CR epitopes and their contribution to antibody repertoire and protection. Although DENV does not undergo antigenic shift and drift to the same extent as some other viruses, antigenic variation in the PF-06873600 E protein can influence sensitivity to neutralization by nAbs [15]. and death [1]. Neutralizing antibody (nAb) responses to dengue are considered important for protection and are characterized as type-specific (TS), directed against only the infecting serotype, or cross-reactive (CR), directed against more than 1 serotype. Studies of immunity to natural infection support a role (1) for TS nAbs in protection against infection with the same DENV serotype and (2) for potent CR nAbs in protection against diverse DENV serotypes [2C4]. It is accepted that, after main dengue infection, potent TS antibodies are elicited against infecting serotype, whereas short-lived CR nAbs are elicited against other serotypes [3, 5]. However, a more recent study exhibited that CR nAbs can persist and increase over time [6]. Takedas live-attenuated tetravalent dengue vaccine (TDV), TAK-003, comprises an attenuated DENV-2 and 3 recombinant viruses with structural premembrane and viral envelope (E) proteins of DENV-1, -3, and -4 cloned into the attenuated DENV-2 backbone [7]. During the phase 3 TIDES study conducted in >20 000 children in dengue-endemic countries, the primary endpoint was achieved with an overall vaccine efficacy of 80.2% against virologically confirmed dengue and 95.4% against hospitalized dengue from any dengue serotype [7]. Vaccine efficacy varied by serotype, and exploratory analysis suggested a lack of efficacy against DENV-3 in baseline seronegative participants, warranting continued monitoring over longer term [7]. TAK-003 elicited tetravalent nAb seropositivity in both baseline seronegative and seropositive participants, with highest titers against DENV-2 [7]. In this study, we characterized nAb responses to TAK-003 for breadth in a subset of baseline-seronegative and seropositive vaccine recipients from a phase 2 study and for serotype specificity in a subset of baseline-seronegative participants from phase 3 clinical studies. METHODS Serum Samples Serum samples were collected during randomized, double-blind, placebo-controlled clinical trials (Supplementary Methods) in accordance with the Edinburgh revision of the Declaration of Helsinki, International Conference on Harmonisation and Good Clinical Practice guidelines, and relevant national and local regulations and requirements. Study protocols were approved by the local internal review boards, and written informed assent or consent was obtained from all participants or their legal guardians. DEN-205 (ClinicalTrials.gov Identifier NCT02425098) compared immune responses in Singaporean adults to single doses of 2 early formulations of TAK-003, termed high-dose (HD)-TDV or TDV [8]. DEN-205 samples included antisera from individuals who were baseline-seronegative (= 10) or seropositive (= 10) to DENV, before vaccination with HD-TDV (= 18) or TDV (= 2). DEN-301 (ClinicalTrials.gov Identifier NCT02747927) evaluated efficacy of 2 doses of TAK-003 against symptomatic dengue fever due to DENV-1C4 in participants aged 4C16 years in the Philippines, Thailand, Sri Lanka, Colombia, Panama, Brazil, Dominican Republic, and Nicaragua [7]. DEN-304 (ClinicalTrials.gov Identifier NCT03423173) was a manufacturing consistency study of 2 doses of TAK-003 in PF-06873600 baseline-seronegative DEN-301 (= 46) and DEN-304 (= 25) adults aged 18C60 years in the United States (VT, PJW, LMT, MR, IE, EH, IL, AB, and DW, 2022, unpublished observations). Dengue Computer virus-2 Depletion Tosyl-activated Dynabeads coupled with pan-flavivirus 4G2 monoclonal antibody were bound with either live DENV-2 (strain 16681) or bovine serum albumin ([BSA] control), blocked with BSA to prevent nonspecific interactions, and then incubated with heat-inactivated vaccine-recipient sera to deplete DENV-2 TS and CR C5AR1 nAbs (Supplementary Methods, Physique 1= 71) collected 30 days after second vaccination from studies DEN-301 (reddish dots) PF-06873600 and DEN-304 (blue dots) were depleted with mock antigen (x-axis) and DENV-2 antigen (y-axis) bound to magnetic beads, and their neutralization titers were measured by dengue RVP assay. Log10 EC50 represents the dilution factor required to neutralize 50% of RVPs. Samples where depletion of anti-DENV-2 antibodies prospects to complete loss of detectable nAbs against DENV-1, 3 and/or 4 (seen as log10 EC50 < limit of detection (LOD) and represented by the open circles) contain a high proportion of cross-reactive antibodies. Samples where.